We show that long-term exposure to a ketogenic diet, fed every other week starting in middle age, reduces midlife mortality and preserves memory in aging C57BL/6 male mice. Similar feeding of a high-fat, low-carbohydrate non-ketogenic diet appeared to have an intermediate effect on mortality, but survival of this Cyclic HF group could not be definitely distinguished from either the Control or Cyclic KD groups. These results might be interpreted in the broader context of the health effects of DR and of segmental DR mimetics such as metformin and rapamycin, and suggest that one or more aspects of a ketogenic diet may similarly act as a segmental DR mimetic. One prior study of KD did not observe a change in survival, but differences included the use of a very-low-protein KD formulation (5% of calories), initiation in young mice (8 weeks old), and substantially shorter lifespans in all groups (Douris et al., 2015). Importantly, all of the diets used here had identical per-calorie protein content. Protein-restricted KD may not produce obesity in mice, but our finding that a normal-protein KD is obesogenic is consistent with prior reports (Borghjid and Feinman, 2012).

Unlike DR, the effect on survival we observed from intermittent feeding of KD was not consistent throughout the lifespan. The mortality in the Cyclic KD group was lowest from 12 to 30 months old, then converged with the Control group. The lifespan-extending fasting-mimic diet (FMD), which involves severe DR 3–4 days per month, showed a similar pattern (Brandhorst et al., 2015). One explanation may be that changes to cellular physiology in older animals, such as to histone acetylation (Peleg et al., 2016), attenuate the response to the specific mechanisms of DR invoked by Cyclic KD. Alternatively, weight cycling could become a counterproductive stress in old age. In addition to the FMD, a small study of monthly cycling on a high-fat diet also appeared to show increased mortality in old age (List et al., 2013). Finally, there could be an accumulated harm that becomes evident only with prolonged intake of HF or KD, such as, perhaps, liver injury. Understanding the mechanism of this potential harm at old age or long duration will help ensure the safety of any translational a

pplications of cycling or ketogenic diets in humans.

In healthspan testing, we found a striking effect of Cyclic KD on memory as well as more modest effects on a broader range of measures. We saw consistent memory improvement in two distinct tasks over 6 months. Effects of KD on activity were mixed, with no change in the open field or running wheels but amelioration of the age-related decline in elevated plus maze activity. Physical performance was consistently not affected, even in those tasks with clear age-related declines in Controls (balance beam training and single-wire hang) and in those with strong neurocognitive elements (e.g., balance beam). Protein intake was similar in both groups, but it is possible that the periodic weight loss associated with the cyclic regimen limited any improvements in motor strength.

Strengths of this study design for evaluation of a ketogenic diet included the careful matching of protein content; a regimen (alternating KD and Control diet weekly) that approximated both body weights and caloric intake to the Control-fed group; a comparison group fed a maximally high-fat, low-carbohydrate but non-ketogenic diet; parallel healthspan and lifespan cohorts; and feeding Control diet to all mice during healthspan testing to avoid acute confounding effects of ketosis or weight changes. This study also had important limitations, including the use of a single strain, single sex, single start age, single non-obese ketogenic diet regimen; lack of a Cyclic HF group in healthspan testing; and lack of a separate pathology cohort. All of these choices increased the power of the primary comparison but left potentially important variables to be explored in future studies. We selected C57BL/6 males because they are particularly prone to lifespan extension with DR, even when started in middle age (Pugh et al., 1999). But as most longevity interventions in mice have shown different responses between sexes (Austad and Fischer, 2016) and strains (Liao et al., 2010), it will be critical to test the effect of ketogenic diet or ketone bodies in other backgrounds. Similarly, starting a ketogenic diet intervention at different ages and/or using a variety of durations could determine if the waning benefit of Cyclic KD late in life is due to increasing age or to increasing duration of treatment. Testing other regimens for feeding ketogenic diets or ketone bodies can ensure that the phenotypes we observed were not due solely to the every-other-week diet cycling and would provide additional data toward the relevant mechanisms at work. Our study cannot exclude that a cyclic non-ketogenic high-fat, low-carbohydrate diet might have similar effects on memory with aging as the cyclic ketogenic diet. Finally, although we collected necropsy data when animals were euthanized for cause, this could be subject to selection bias and cannot describe age-matched incidence of pathologies as a dedicated pathology cohort could have.

The possible mechanisms of KD in longevity (and cognition) include effects of the low-carbohydrate, high-fat diet composition as well as activities of BHB itself. The former include reduced insulin and IGF signaling, reduced protein synthesis, and suppression of TOR activation. Our gene expression studies helped to distinguish which mechanisms are shared in common with a high-fat, low-carbohydrate diet, and which are unique to KD. KD has been associated in the liver with downregulation of genes involved in fatty acid synthesis and upregulation of fatty acid oxidation genes (Douris et al., 2015), including PPARα target genes (Badman et al., 2007). Indeed, PPARα activation is a master switch that is required to fully activate the response to fasting or a ketogenic diet (Badman et al., 2007). A “Western” high-fat diet (not low-carbohydrate) has a distinct profile from KD, with upregulation of fatty acid synthesis and gluconeogenesis genes and downregulation of fatty acid oxidation (Kennedy et al., 2007). Our data show that a high-fat, low-carbohydrate diet is remarkably similar in transcriptional profile to KD. Both suppress fatty acid synthesis, glucose metabolism, and protein synthesis, all of which might provide a mortality benefit common to the cyclic (non-obese) HF and KD used here. The potent upregulation of PPARα target genes is unique to KD and might provide one clue to the mechanisms of the incrementally larger effect on survival of Cyclic KD. PPARα activation has been suggested as a calorie restriction mimetic (Barger et al., 2017), but its effects on aging phenotypes have not been deeply explored.

Direct gene expression effects of histone beta-hydroxybutyrylation are a tempting mechanism to link BHB to the upregulation of PPARα target genes that we observed—both acutely after 1 week on KD and persistent in old Cyclic KD mice several days into a Control diet week. Indeed, Xie and colleagues reported that BHB-modified H3K9 marks PPARα target genes and is associated particularly with such genes upregulated (but not downregulated) by prolonged fasting (Xie et al., 2016). It is not yet known if histone beta-hydroxybutyrylation is merely a marker of transcriptional activation or is an effector of activation. Our data would fit well into a model in which BHB itself provides additional modulation of the baseline transcriptional program of a high-fat, low-carbohydrate diet. Whether these transcriptional changes directly modulate the effects of Cyclic KD on mortality or memory remains to be tested. Other models are also plausible. For example, deacetylase inhibition is also associated with many of the effects we observed for Cyclic KD, including preservation of memory (Penney and Tsai, 2014), amelioration of chronic heart remodeling (Morales et al., 2016), and even activation of PPARα (Montgomery et al., 2008).

We have shown that long-term exposure to BHB through a ketogenic diet reduces midlife mortality and preserves memory in aging normal mice, while activating a distinct transcriptional program involving PPARα target genes. Further work might determine if BHB itself is a segmental mimetic of DR and could provide candidates for translational therapies of syndromes of aging through either administration of ketone esters (Veech, 2014) or, with a thorough understanding of its downstream signaling mechanisms, activation of BHB’s specific signaling effects. Future work to understand the nature of the persistent effect on memory in particular may lead to therapies to promote cognitive resilience to dementia or illness-associated delirium.